This proposal aims to provide preclinical data in support of a novel therapy for epilepsy in Tuberous sclerosis complex (TSC), a disorder that includes early life epilepsy, mental retardation and autism. We propose that bumetanide, an inhibitor of the chloride cotransporter NKCC1, will be effective for the prevention of seizures in TSC alone or in combination with a canonical GABA agonist phenobarbital or the recently approved anticonvulsant vigabatrin. Our recent publication shows that NKCC1 is overexpressed in human TSC tissue, as well as in another cause of refractory epilepsy, focal cortical dysplasia Type IIb (FCD IIb). Increased NKCC1 causes enhanced intracellular chloride, and this impairs inhibitory actions of GABA receptor activation by causing it to mediate depolarization and excitation. We have recently generated a novel Tsc1cc Nestin-rtTA+ tet-OP-cre+ mutant mouse model that is epileptic and shows NKCC1 overexpression and depolarizing GABA responses. Aim 1 will use this mouse model to further text the efficacy of bumetanide alone or in combination with phenobarbital or vigabatrin in cortical slices in vitro and by treatment in vivo using EEG recordings. Aim 2 will examine the effects of bumetanide alone and in combination with phenobarbital or vigabatrin on GABA responses in brain slices acutely prepared from human TSC and FCD IIb surgical biopsy tissue. The proposed work represents a new approach in studying the mechanisms of epileptogenesis in TSC and has the potential to generate an adjuvant novel mechanism-based anticonvulsant strategy for seizure control in TSC patients. It will also provide proof-of-principle for this mechanism, as well as preclinical efficacy in a TSC mouse model with clinically relevant outcomes. Finally our proposal includes target validation as well as mechanism in human tissue. These are critical elements of a path to first-in-human trials for this compound in TSC, and may also extend to FCD IIb.